The National Cell Repository for Alzheimer's Disease: A genetic researcher's 'savings and loan'

The National Cell Repository for Alzheimer's Disease: A genetic researcher's 'savings and loan'

You might think of the National Cell Repository for Alzheimer’s Disease (NCRAD) as a savings and loan association for Alzheimer’s disease (AD) researchers. Its assets are human cell lines and DNA samples, plus a database containing family histories, demographics, and participants’ medical information. NCRAD “lends” its assets to researchers, who “repay” the loan by adding to the growing body of knowledge about AD. Funded by the National Institute on Aging (NIA), NCRAD is staffed and managed by the Indiana University School of Medicine in Indianapolis.

When asked to describe a typical day at NCRAD, Tatiana Foroud, Ph.D., Director, says “There really is no such thing for us. In fact, some days may seem a little frantic!” Dr. Foroud should know. She is the lead investigator at NCRAD and became director in 2004. She gives the following example to illustrate how flexibility is needed during any given day:

“When a staff member learns that a study participant who agreed to donate his or her brain to NCRAD has died, we have to shift gears completely. Brain tissue is very fragile and highly valued for research. Our ready response team must move quickly to help the family arrange an autopsy, wherever the donor might be; find someone qualified to remove the brain; get the organ to a neuropathologist for analysis; and arrange shipment to NCRAD, all before significant deterioration occurs. Analysis of this tissue is the only way to confirm or disprove a diagnosis of AD. Chances for success are greatly enhanced when the donor or family has pre-planned the autopsy and the details are in place at NCRAD.”

On a “Normal” Day

During more routine days, NCRAD receives and processes blood samples from as many as 15 different sources. They arrive in tubes from participating Alzheimer’s Disease Centers (ADCs) and other approved sites across the country. Samples also may be hand-delivered by staff members returning from site visits. The study coordinator assigns each one an ID number and removes other identification. Only descriptive information, such as gender and year of birth, accompanies each sample from there.

Data collection

A variety of information is obtained about each person who provides a blood sample. Some NCRAD collections include corresponding family history and medical information for each sample, while others contain only demographic and diagnostic information. The different collections housed at NCRAD are distinct based on data collected, family history, age of onset, and diagnostic requirements. Samples are cataloged by a variety of criteria, such as age of onset and certainty of diagnosis.

In addition to samples from a specific population type, a researcher may want samples from subjects without signs of dementia, who have no closely related family members with AD. Comparing the DNA of these “controls” to that of people with AD helps researchers understand which parts of the DNA (or genes) contribute to or reduce the risk of AD.

Information is often updated, so that researchers can compare a participant’s initial diagnosis with any new information and finally with autopsied brain tissue. This information also helps to ensure that researchers receive the kinds of samples they need for a particular study, based on their study criteria, which may include diagnostic status (e.g., confirmed AD, probable AD, possible AD, or another type of dementia), race, ethnicity, and/or age of disease onset. Staff members enter this vital information into a secure database, where only ID numbers identify donors. Strict donor confidentiality and anonymity are maintained.

A researcher requests cell line samples to obtain an unlimited source of DNA or to perform analyses requiring intact cells. A researcher may request frozen DNA samples when an unlimited supply of DNA isn’t needed. New technologies allow scientists to examine a single position of interest on a DNA strand or thousands of positions.

Lab work

Next, the sample goes to the lab, where the coordinator applies a unique barcode to the tube. The ID number and barcode help track the samples at various stages of processing while maintaining donor privacy and anonymity throughout the process.

Blood samples are spun down into three layers—red blood cells, plasma, and white blood cells. The white blood cells are extracted to produce cell lines and DNA, which carries genes that determine inherited characteristics, such as the risk of AD.

Information for researchers

The NCRAD Cell Bank Advisory Committee must approve requests for samples. Typically, approved research focuses on aging, diseases associated with aging, or the etiology, pathogenesis, diagnosis, treatment, or prevention of AD. Research proposals must outline:

Intended use of the samples

Description of the research project

Type and number of samples requested

Name of the Principal Investigator and others who will use the samples

Any grant or contract supporting the work

Applicants must agree to protect the privacy of donors and share the results of genetic analyses.

Production of cell lines

To produce cell lines, a lab technician places the white blood cells from the donated sample into a flask with a solution that promotes cell division. The new solution is incubated at human body temperature (37°C) for several weeks and then transferred to two larger flasks. Cell division continues until desired quantities of cells are achieved.

Then the cells are put into “cryo-vials” (about 10 million cells per vial), with a “cryo-preservative.” These vials are cooled in a controlled-rate freezer. Slow cooling prevents cell damage. Finally, the frozen vials are stored in a tank filled with liquid nitrogen, which takes them down to -193.3°C.

This process provides a stock of cells for future use. These cells can be thawed at any time and cell division will resume. In this way, more cells can be grown to replenish a cell line.

Production of DNA samples

Some white blood cells are washed and spun at high speed, causing them to form a pellet. The cell pellet is placed in solution with an enzyme that breaks down the cells but leaves DNA intact. After processing, the DNA sample is stored at -80ºC.

Participant contact

Each year, NCRAD re-contacts donors participating in the original NCRAD collection to update information such as family deaths, along with cause of death, and information about onset of AD, another type of dementia, or memory loss. Kate Kreiner, a NCRAD Clinical Research Specialist, works closely with these families. She conducts cognitive assessments of participants by administering a 30-minute questionnaire via telephone. Sheexplains: “The battery of tests is a way to quantify performance over time, but not to find out if you have AD. In families, it’s important also to know who isn’t having memory problems or getting the disease.”

Delivery of specimens

A researcher who is interested in obtaining samples from one of the collections housed at NCRAD works closely with one of the NCRAD study coordinators. Requests are reviewed by NCRAD’s Cell Bank Advisory Committee. Upon approval, a NCRAD coordinator sends the request to the laboratory, where a technician retrieves, thaws, and prepares samples for shipment. DNA can withstand some delay in delivery, but cell lines must arrive promptly to remain viable.

An unsurpassed resource

NCRAD’s collections are unique because:

While other cell repositories focus on samples from separate, unrelated individuals, NCRAD collects and stores samples and information from families, including both members diagnosed with AD and others diagnosed as AD-negative. According to Dr. Foroud, “Having specimens from families with established patterns of AD improves researchers’ chances of finding genes with specific mutations in common.”

While many cell repositories don’t follow up with donors, NCRAD updates its donor information annually.

The number of samples a researcher can request is, in theory, limited only to the amount of sample material available (larger requests may be evaluated more closely before approval). This ready availability of samples is an advantage for researchers conducting studies that require many samples from one source, along with uniform background data.

NCRAD coordinators work closely with researchers requesting samples to ensure they get precisely what they need to meet their unique research requirements.

Proven value

To date, researchers around the world who use NCRAD resources have published more than 120 scientific papers. Among them is the discovery in the 1990s of an allele of the ApoE gene—ApoE4, the first gene to be linked with increased risk of late-onset AD (LOAD).

Researchers are using NCRAD resources to track down other genetic factors in AD. Many people with the ApoE4 allele don’t get AD, suggesting that other genes, as well as environmental and lifestyle factors, may increase or protect against the risk of AD, but these factors remain to be discovered. NCRAD samples also were used to understand the role of PS1, PS2, and APP genes as the genetic causes of familial AD (AD diagnosed before age 65).

“NCRAD is a unique and critically important resource for advancing our understanding of the mechanisms and causes of AD. As researchers continue to uncover more promising information about genetic factors associated with increased risk of AD, we’ll be able to refine our efforts toward possible future treatment and prevention therapies,” says Marcelle Morrison-Bogorad, Ph.D., Director, Neuroscience and Neuropsychology of Aging Program, NIA.

What NCRAD doesn’t do

NCRAD doesn’t use blood samples to test for AD, but only to obtain cell lines and DNA for research. Because of this research focus, NCRAD doesn’t provide certain services, such as genetic counseling to donors. According to Teresa Evans, a NCRAD Clinical Research Specialist: “NCRAD will refer families inquiring about this service to genetic counselors who have a background in psychology and expertise in discussing genetic test results with clients. Our work is usually done over the phone, but families who feel that they need counseling should receive these services from these experts, face-to-face.”

Contributing to NCRAD

NCRAD can meet growing demand for biological research samples only with the help of volunteer families who meet inclusion criteria.

According to Richard Mayeux, M.D., Principal Investigator for the NIA-funded Alzheimer’s Disease Genetics Study, “These samples are tremendous assets for researchers who need large datasets for genetic analysis. Because the samples are so well-characterized, they provide a valuable bank for scientists searching for risk factor genes. We’ll be able to draw upon these reserves for a long time.”

Participation in NCRAD is totally voluntary. Criteria for participation in the two large NCRAD collections include:

Original NCRAD collection:

Families with at least two living, blood-related relatives with symptoms of early-onset (familial) AD, late-onset AD (LOAD), or dementia.

LOAD collection (Alzheimer’s Disease Genetics Study-ADGS):

At least three people in a family must meet these requirements:

The “proband”—a person with AD—must have disease onset after age 60 and agree to be seen, evaluated, and diagnosed at an NIA-funded ADC or other approved institution.

A second family member must have disease onset after age 60 and be a sibling of the proband.

A third family member must be related to the proband and either unaffected by AD and at least 60 years of age, or diagnosed with AD after age 50.

Once enrolled, families or individuals can discontinue their participation at any time. Volunteers receive no payment for their participation, but know that their generosity may contribute to a cure. There is no cost for participants. All information remains confidential.

As whole genome association studies evolve, the expanded NCRAD collection will play an important role.

National Cell Repository for Alzheimer’s Disease (NCRAD)
Indiana University School of Medicine
Department of Medical and Molecular Genetics
410 West 10th Street, HS 4000
Indianapolis, IN 46202-3002

NCRAD by the numbers*

*All numbers are approximate totals compiled since 1990.

28,000
Number of biological samples NCRAD has sent to AD researchers

7,000
Number of participants who have provided blood samples

900
Number of families participating in the NCRAD collection

800
Number of families participating in the LOAD (ADGS) collection

560
Number of NCRAD participants who have had a brain autopsy

430
Number of participants who pro-vided samples and had an autopsy

380
Number of participants with a sample with confirmed AD via autopsy

120
Number of scientific papers published using NCRAD samples

75
Number of leading researchers using NCRAD samples.

50
States where participants live, plus Puerto Rico (and 7 countries)

Update: the Alzheimer’s Disease Genetic Study (ADGS)

Since 2002, the NIA has funded the ADGS, a nationwide effort to collect genetic material from families affected by late-onset Alzheimer’s disease and to make it available to researchers. The goal of ADGS planners is to recruit 1,000 families that meet the criteria for the study. These families are being recruited by selected ADCs across the country. Recruitment began in 2003. So far, more than 1,000 families have inquired, almost 900 are eligible for the study, and more than 800 have donated samples. The enrollment phase of the study is expected to be completed in 2008. Analysis of these genetic samples will continue for years to come.